What Are the Microscopy Techniques in Observing Tissue Culture?

Microscopy is the technique used when producing visible images of minute details or structures. This can be accomplished with the use of a magnification tool such as a microscope. As microscopes evolve, so did microscopy.

Here are the known microscopy techniques in observing tissue culture:

Brightfield Microscopy

Of all the microscopy techniques that utilize light, brightfield microscopy is known to be the simplest. The sample illumination is through white light that is transmitted, such as the light observed above but illuminated from below. Aside from its very simple principle, it is also very easy to set up since you’re only going to use a very basic equipment. There’s also no need for sample preparation so you can actually view live cells. However, they offer very low contrast for majority of biological samples. Moreover, because it’s simple, it has low resolution because of the out-of-focus or blurry material.

You can, however, enhance this technique. You may minimize the size of the source of light through the condenser aperture. Take note, though, that this procedure can also reduce the resolution. You can also use polarized or colored filters into the source of light. This will highlight features that are not seen under white light.

Oblique Illumination

Oblique illumination can sometimes be referred to as sideways illumination. You either cover a portion of the source of light to project asymmetrical lighting or you can simply shine an external source of light sideways into the sample. This provides a 3D image of the specimen and can also highlight features not seen by the naked eye. A more contemporary technique similar to this process is known as Hoffman’s modulation contrast. Inverted microscopes used for studying cell cultures make use of this system.

Like brightfield technique, you only use a basic equipment sop there’s no need for any complicated setup. There’s also no requirement for sample preparation. Nevertheless, its limitations are similar to brightfield method as it has very low contrast and resolution. It cannot produce sharp images.

Rheinberg Illumination

This is a unique version of the dark field illumination. It was introduced by Julius Rheinberg. Colored but transparent filters are attached before the condenser. This is so, so light rays belonging to a higher aperture will appear differently colored than light rays having lower aperture. A good example can be a specimen placed in a blue background. Because of this technique, the object will appear yellow. You can also probably obtain other color combinations, but the overall effectiveness can vary.

Dark Field Microscopy

A light source carefully aligned is being employed when it comes to dark field microscopy. This is to reduce the amount of light transmitted directly into the image. Only the collected light can be scattered by and into the sample. You can confine the light through the use of rings. Dark field microscopy can make transparent objects present in the sample very visible. It is also very convenient to set up and there’s no sample preparation needed. However, if offers low light intensity for the final image as well as very low resolution.

Phase Contrast Microscopy

This is a very popular technique that can showcase differences of refractive index as contrast differences. It was Fritz Zernike, a Dutch physicist, who was able to develop such method during the 1930s. His discovery even earned him an award in 1953—the Nobel Prize for Physics. For example, the cell’s nucleus will appear dark against the cytoplasm surrounding it. There’s superb contrast, only that you cannot use such technique with thick materials. Normally, a halo will be formed surrounding the small object. This can obscure the detail.

The system is composed of a round annulus found in the condenser. This can produce conical light, which is placed over a ring of similar size found in the phase-objective. Each of the objectives requires another size of ring. So a different condenser setting is selected depending on the objective used. The objective’s rings possess certain distinct properties. First, it can reduce the intensity of direct light. Second, it develops a fake phase difference that’s about ½ of the actual wavelength. When physical properties of the direct light are altered, there occurs interference in the diffracted light. This results to phase contrast images.

Fluorescence Microscopy

If particular compounds are illuminated with light of high energy, the specimen can emit light of a much lower and different frequency. This phenomenon is more commonly referred to as fluorescence. Depending on the chemical makeup of the specimen, they may exhibit their own autofluorescence image.

One of the microscopy techniques in observing tissue culture, this method is highly essential. It can be very sensitive that it can even detect single molecules. Laboratories make use of fluorescent dyes for staining different cell cultures.